The tumor suppressor protein p53 (the guardian of the genome) is a transcription factor that plays a key role in preventing cancer development. In malignant tumors, hot-spot mutations of p53 usually cause p53 misfolding and functional loss in more than 50% of human cancers. Successful development of novel mutant p53-reactivating anticancer drugs is a promising strategy for cancer treatment. There are several approaches to identify small molecules that target misfolded p53, including rational design and screening of chemical libraries. Thus, a number of p53-activating compounds have been identified from chemical libraries using protein- and cell-based screening assays. However, screening methods have yet to deliver a small molecule compound that rescues oncogenic p53 mutants by directly binding to its core domain, and further work is required to achieve this using improved drug screening platforms. Several experimental techniques are available for in vitro and in cellulo evaluation of protein folding, but none of them to date can be optimally extended to imaging assays in intact living subjects. The latter can characterize and quantify these biological processes in living subjects within realistic and relatively undisturbed confines of cels that are also present in the midst of fully functional and intact whole-body physiological environments. This therefore allows additional consideration of pharmacodynamics and pharmacokinetics of test drugs. Hence, in this project we will address the development and characterization of a new generalizable biosensor that exploits an in vivo protein-fragment-assisted complementation assay (PCA) based on a split bioluminescence reporter (synthetic humanized renilla luciferase [hRluc]) complementation technology, to pre-clinically image protein folding/misfolding in intact living subjects. The immediate goals of this exploratory research are: (Aim 1) to initially establish and comprehensively validate robust generalizable molecular engineering rules for building a split reporter-based imaging folding biosensor using split hRluc with the circularly permuted variants of enhanced green fluorescent protein as the test proteins; and (Aim 2) to then use these rules to construct a novel p53 folding biosensor for detection of conformational changes of the p53 molecule in living mice. For this we will construct and initially test the biosensor within variable p53-status cellular environments in cell culture; use the sensor in cell culture to study and image the rescue of p53 function by small molecule drug therapy of misfolded p53; and image and validate in living mice the biological functioning of the sensor. The long-term goals are to develop novel molecular imaging-based high throughput screening (HTS) platforms using the p53 folding sensor to accelerate the search for better drugs targeting p53 misfolding, which may ultimately lead to the prevention of tumor development and is a major goal in cancer research. To that aim, the proposed work will provide for the first time an important new molecular imaging assay tool to evaluate pharmacological reactivation of misfolded p53 in living subjects.